This application relates to a new mutants of the enzyme dihydrofolate reductase, and to the use of these mutants as selectable markers and for gene therapy to produce drug resistant bone marrow or peripheral stem cells.
Dihydrofolate reductase (DHFR, 5,6,7,8-tetrahydrofolate:NADP+oxidoreductase, EC 1.5.1.3) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, an essential carrier of one-carbon units in the biosynthesis of thymidylate, purine nucleotides, serine and methyl compounds. DHFR is an essential enzyme in both eukaryotes and prokaryotes.
In rapidly dividing cells, the inhibition of DHFR results in the depletion of cellular tetrahydrofolates, inhibition of DNA synthesis and cell death. Because of this, folate analogs which inhibit DHFR, for example methotrexate (MTX), are used as antineoplastic agents. The utility of xe2x80x9cantifolatexe2x80x9d treatments of this type is limited by two factors. First, tumor tissues may rapidly develop resistance to the antifolate, rendering the treatment ineffective. Second, the treatment may be toxic to rapidly dividing normal tissues, particularly to bone marrow or peripheral stem cells.
International Patent Publication No. W094/24277, which is incorporated herein by reference, discloses mutant forms of human DHFR which have increased resistance to inhibition by antifolates used in therapy includi MTX. The specific mutants disclosed differ from wild-type human DHFR as a result of a single mutation occurring at amino acid 15, 31 or 34.
Mutations at amino acid 22 of human DHFR have also been shown to reduce the sensitivity of the enzyme to antifolate inhibition. Ercikan et al., in Chemistry and Biology of Pteridines and Folates, J. E. Ayling, ed., Plenum Press (1993). In these mutants, the amino acids isoleucine, methionine, phenylalanine and tyrosine are substituted for the leucine of the wild-type enzyme.
The present invention provides new mutant forms of human DHFR which have properties superior to the previously disclosed mutants. In particular, the present application is addressed to mutant forms of human DHFR which have mutations at both amino acid 22 and amino acid 31. Preferred mutant forms within the scope of the invention are Ser31Tyr22, Ser31Phe22, Gly31Tyr22, Gly31Phe22, Ala31Tyr22 and Ala31Phe22.
The mutant DHFR of the invention may be used as a selectable marker. Thus, an aspect of the present invention is a method of selecting among clones for the introduction of a non-selectable gene comprising the steps of
(a) inserting the non-selectable gene into a DNA vector comprising DNA encoding a mutant form of human dihydrofolate reductase which differs from wild-type human dihydrofolate reductase at both amino acid 22 and amino acid 31, wherein the mutant form has an amino acid with a larger volume side chain than leucine at amino acid 22 and an amino acid having a smaller volume, more hydrophilic side chain than phenylalanine at amino acid 31;
(b) introducing the vector containing the non-selectable gene into cells of a type in which the non-selectable gene and the mutant form of dihydrofolate reductase are expressed; and
(c) selecting cells which are resistant to inhibition by antifolates.
The mutant DHFR of the invention may also be used to modify the genome of human cells, particularly bone marrow cells or peripheral blood stem cells to render them resistant to chemotherapy using antifolate agents. Thus a further aspect of the invention is a method for gene therapy comprising the steps of:
(a) obtaining hematopoietic cells from a human patient;
(b) transducing into the hematopoietic cells an expressible mutant form of human dihydrofolate reductase which differs from wild-type human dihydrofolate reductase at both amino acid 22 and amino acid 31, wherein the mutant form has an amino acid with a larger volume side chain than leucine at amino acid 22 and an amino acid which having a smaller volume, more hydrophilic side chain than phenylalanine at amino acid 31; and
(c) returning the transduced cells to the human patient.